Abstract
MicroRNAs (miRNAs) are small noncoding RNAs that regulate the stability and expression of target RNAs in a sequence-dependent manner. Identifying miRNA-regulated genes is key to understanding miRNA function. Here, we describe an unbiased biochemical pulldown method to identify with high-specificity miRNA targets. Regulated transcripts are enriched in streptavidin-captured mRNAs that bind to a transfected biotinylated miRNA mimic. The method is relatively simple, does not involve cross-linking and can be performed with only a million cells. Addition of an on-bead RNase digestion step also identifies miRNA recognition elements (MRE).
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Acknowledgements
We thank Jingmin Jin and Larry McReynolds for their sequencing expertise, Rory Kirchner, Oliver Hofmann and Winston Hide for their bioinformatics expertise, and members of the Lieberman lab for critical discussions. S.M.T. was supported by the Department of Defense (DOD) Breast Cancer Research Program (BCRP).
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Tan, S.M., Lieberman, J. (2016). Capture and Identification of miRNA Targets by Biotin Pulldown and RNA-seq. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 1358. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3067-8_13
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DOI: https://doi.org/10.1007/978-1-4939-3067-8_13
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3066-1
Online ISBN: 978-1-4939-3067-8
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